1. Field of the Invention
The present invention relates to a fluorescence detecting device to be used for observation and/or measurement of biological tissues and cells in the medical and biological fields.
2. Description of the Related Art
The fluorescent detecting device is usually used for detecting proteins and genes marked with a fluorescent marker on the biological tissues and cells in the medical and biological fields. Since observation of extremely weak fluorescence emitted from one molecule of a fluorescent dye has been required in recent years, further improvements in absolute brightness level and S/N ratio are desired.
FIG. 1 shows a basic construction of a conventional epi-illumination fluorescence microscope as a representative example of the fluorescent detecting device. In FIG. 1, the reference numerals 1 denotes a light source such as a mercury vapor lamp and the like, 2 denotes a collector lens, 3 denotes an aperture stop, 4 denotes a field stop, 5 denotes an excitation filter set, 6 denotes a specimen, 7 denotes a dichroic mirror, 8 denotes an objective, 9 denotes a stage, 10 denotes an absorption filter set, 11 denotes a beam splitter, 12 denotes a photography lens, and 13 denotes an observation optical system comprising an imaging lens and an ocular. While the luminous flux projected out of the light source 1 is condensed with the collector lens 2 and passes through the excitation filter set 5 via the aperture stop 3 and field stop 4, the light beam excitation light is converted into a luminous flux having a high intensity at only a desired excitation wavelength for the specimen 6. The luminous flux after passing through the excitation filter set 5 is reflected at the dichroic mirror 7 and irradiated to the specimen 6 on the stage 9 through the objective 8. The fluorescence emanated from the specimen 6 passes through the dichroic mirror 7 via the objective 8 and, after eliminating the light in unnecessary wavelength regions such as the illumination light with the absorption filter set 10 having a high transmittance at the desired fluorescence wavelength band, is guided to the photography lens 12 and observation optical system 13.
FIG. 2 and FIG. 3 show transmittance spectra of the excitation filter set 5 and absorption filter set 10, respectively, in the epi-illumination fluorescence microscope, wherein one kind of the fluorescent dye and two kinds of the fluorescent pigments are used in the experiments shown in FIG. 2 and FIG. 3, respectively. In the graphs, Ab1, Ab2 and Ab3 show transmission bands of the excitation filter sets 5, E1, E2 and E3 show transmission bands of the absorption filter sets 10, and F1, F2 and F3 show fluorescence emission bands. Since the excitation light arriving at the detector causes background noises that deteriorate S/N ratio to give a fluorescence image with low contrast, the transmission bands of E1 and Ab1 should not be almost overlapped with each other. These conditions are the same with respect to the transmission bands of E2, E3 and Ab2. While the wavelength where the transmittance spectrum of the excitation filter set 5 and the transmittance spectrum of the absorption filter set 10 cross with each other is termed a cross-over wavelength, the point X1 in FIG. 2, and the points X2, X3 and X4 correspond to this cross-over wavelength. Therefore, the S/N ratio of the fluorescence detecting device is determined by the width of the transmission wavelength band of the absorption filter set 10 and transmittance of each filter set at the cross-over wavelength. Since most of the fluorescent dye have a fluorescence intensity peak at a wavelength very close to their excitation wavelength, it is crucial that the transmission wavelength bands of the excitation filter set and absorption filter set are very close with each other.
An epi-illumination laser fluorescence microscope making use of a laser light source has been practically used as a fluorescence detecting device. Since the wavelength of the light source is almost monochromatic and a wavelength that can effectively excite fluorescence with a desired wavelength is selected, no excitation filter set is needed in this device. The cross-over wavelength of the epi-illumination fluorescence microscope corresponds to the laser wavelength. The S/N ratio is determined by the width of the transmission wavelength band of the absorption filter set, its distance from the laser wavelength and transmittance of each filter set at the laser wavelength.
The S/N ratio of the fluorescence detecting device is largely influenced by the spectroscopic characteristics of the optical filters in the excitation filter set and absorption filter set. When the excitation light transmission wavelength band of the excitation filter set is made to come sufficiently close to the fluorescence transmission wavelength band of the absorption filter set while suppressing transmittance at the cross-over wavelength, detection with a high S/N ratio is realized along with enabling to obtain a sufficiently bright florescence image without intensifying the excitation light by efficiently detecting florescence, thereby allowing damages to the specimen and fading of the specimen due to the excitation light to be reduced. However, since the optical filter actually involves production errors, the transmission wavelength band of the excitation filter set is often separated from the fluorescence transmission wavelength band of the absorption filter set more than is necessary in order to prevent leaky illumination light at the cross-over wavelength even when the quality of the optical filter is a little distributed. Accordingly, obtaining a fluorescent image with a high S/N ratio has been difficult.
Examples for solving foregoing problems for realizing a fluorescence detecting device with a high S/N ratio are found in Japanese Unexamined Patent Publications No. Hei 5-188299 and No. Hei 9-15171. Japanese Unexamined Patent Publication No. Hei 5-188299 discloses an epi-illumination fluorescence microscope that always ensures an optimum observation of the fluorescence image without being affected by the production errors of the filter and conditions of the specimen, wherein the excitation filter and absorption filter composed of interference filters are supported in a rotatable manner around an axis being perpendicular to the optical axis, and the filters are cooperatively rotated with each other. A method for continuously changing the wavelength width and wavelength of the excitation light is disclosed in Japanese Unexamined Patent Publication No. Hei 9-15171, wherein the excitation filter is constructed by assembling an interference filter for transmitting long wavelength light and an interference filter for transmitting short wavelength light so that respective filters are able to rotate around an axis being perpendicular to the optical axis to form a tunable excitation light source.
Either methods disclosed in the patent publications above are making use of a phenomenon in which the transmission wavelength band of the interference filter continuously shifts toward a longer wavelength or shorter wavelength depending on the incident angle. The transmission wavelength band of the excitation filter is allowed to come close to the transmission wavelength band of the absorption filter while suppressing leaky light at the cross-over wavelength, because the excitation filter and absorption filter composed of interference filters are adjusted by allowing them to rotate around an axis perpendicular to the optical axis.
However, although characteristics of the filters are most effectively utilized in the method disclosed in Japanese Unexamined Patent Publications No. Hei 5-188299 and No. Hei 9-15171, the limits determined by the filter characteristics can be never surmounted. It is also evident that rising-up of the transmittance from the transmission band to the reflection band of the interference filter generally lose its steepness as the interference film is inclined from a plane perpendicular to the optical axis. FIG. 4 shows the characteristics of the interference filter, indicating the change of the transmittance spectrum against the tilt angle of the absorption filter against the plane perpendicular to the optical axis. As is evident from the graph, the S/N ratio may adversely affect the S/N ratio contrary to our expectation depending on the tilt angle of the filter and fluorescence emission wavelength band in the methods described in Japanese Unexamined Patent Publications No. Hei 5-188299 and No. Hei 9-15171. A considerable proportion of the important and currently available fluorescent dye have fluorescence intensity peak wavelengths very close to their the excitation wavelength bands. A detecting device capable of obtaining a bright image with as high a S/N ratio as possible is desired, because the conditions of the fluorescence detection have became very severe in recent years in the fluorescence observation of living cells, such that the intensity of the excitation light should be as weak as possible in order to reduce damages on the living specimen and fading of the specimen.